Part:BBa_M50447:Experience

Demetri's glycerol stock of DNA in E. coli: barcode number 0133018612

Tom's glycerol stock of DNA in E. coli: barcode number 0133024507

plasmid strain: glucose+insulin

We experienced Negative functionality for our part.

Methods:

DNA Preparation and Colony Prep

Our DNA construct was synthesized by Atum. Once received, we transformed this construct into yeast using a previously established protocol using -URA3 as a selection marker in yeast extract-peptone-dextrose (YPD) media.19 After 48 hour growth at 30℃, we isolated a single clone and streaked it onto a new YPD plate with the same -URA3 selection. This single yeast clone was used to create liquid cultures for all subsequent experimentation.

Glucose Induction Experiment

Yeast liquid cultures from our previously prepared single clone were grown overnight in 2% raffinose. Absorbance measurements at 600 nm were taken following the overnight incubation. Cells were diluted to an optical density of 0.1 in YPD media and mixed with glucose to start the induction. Because we wanted to maintain a constant carbon source concentration of 2%, we added raffinose as we varied glucose. Seventeen concentrations of glucose were tested ranging from 0 mM (0%) to 111 mM (2%). Increments of 5 mM were used for the range from 0 mM - 50 mM and increments of 10 mM were used from 60 mM - 111 mM. Five separate controls were also run. This included three blanks; one with just YPD, one with YPD plus glucose as the carbon source, and one with YPD plus the identified ideal non-glucose carbon source; one control with cells (0.1 O.D.) but no carbon source; and one sample with cells taken from the previous liquid culture. 200 μL of each prepared induction mix was added to a separate well in a clear bottom 96-well plate. Each induction mix was run in triplicate on the plate and incubated for 24 hours. The plate was incubated on a shaker inside the plate reader at 200 RPM and temperature controlled at 30℃. GFP expression was quantified with two different fluorescent reads on the plate reader, one with an excitation/emission of 480 nm / 520 nm and one with an excitation/emission of 395 nm / 509 nm. Fluorescent reads were taken every 30 minutes during the 24 hour induction. Absorbance at 600 nm was also measured every 30 minutes with the fluorescent reads to monitor cell growth.

Western Blot

The protocol for the Western Blot was obtained from our laboratory manual.19 We used a rabbit anti-His tag antibody directed against the His-tag on our insulin protein as a primary antibody (1:1000 dilution) and used a goat anti-rabbit secondary antibody (1:1000 dilution) as our secondary antibody. The secondary antibody was conjugated to HRP, which allowed us to visualize protein production after adding Opti-4CN chromogenic substrate.

IV. Results

GFP Expression Assay

We knew that our plasmid sequence was correct because it was sequence verified by Atum before it was sent out. From here, the goal of our initial prep experiments was to confirm that our plasmid had actually entered our yeast cells, in other words to confirm that our transformation had worked correctly. Our experimental plate, which was treated with kanamycin, from the transformation showed colonies after a 48 hour incubation at 30℃ whereas no colonies appeared on our negative control. Furthermore, a single isolated colony grew up properly when it was streaked onto a new plate with kanamycin, which showed that our transformation was successful. Once we were certain that our plasmid had properly entered our yeast cells and was the correct sequence, we proceeded to perform our 24 hour glucose induction experiment to measure the effect of changing glucose concentration on insulin production. We measured change in insulin production via GFP expression since EGFP was linked to the insulin gene via a T2A peptide under the same promoter. Yeast cells were grown in 2% raffinose overnight and then induced with varying levels of glucose ranging from 0 mM - 111 mM (2%). Our 0 mM glucose sample was our negative control, in which we expected no GFP expression to be induced. We considered a sample treated with 2% glucose to be our positive control because the previous iGEM team reported that 2% glucose was a sufficient amount of glucose to induce GFP expression.4 However, our glucose induction experiment did not show a statistically significant difference between GFP expression for samples of high glucose concentration (2%) and samples with no glucose at all (our negative control) (See Figure 2). All of our samples showed a similar decrease in GFP expression (measured in Relative Fluorescence Units (RFU)). Importantly, our blank sample, which was comprised of only media, did not experience any decreases in GFP expression, suggesting that the decreases in GFP expression were correlated with cell growth. That our “induced” cells and our uninduced cells experienced no significant difference in GFP expression over time, therefore, suggests that our treated yeast cells were not induced by glucose at all, leading us to believe that our promoter did not turn on given the presence of glucose.

see figure 2.

Scatter plot showing GFP expression over time with different concentrations of glucose induction. Glucose induction at concentrations of 0, 25, 50, 80, 100, and 110 mM are shown along with additional controls. Time is shown on the x-axis and GFP expression in relative fluorescence units is shown on the y-axis. An excitation filter of 480 nm with an emission filter of 520 nm was used for the data in this graph. Black data points represent glucose induced samples and colored data points represent controls. Error bars are +/- S.D.

Western Blot

We hypothesized that the T2A peptide could have been sequenced erroneously, suggesting that our promoter might have worked properly, yet the ribosome could not translate the GFP gene. Therefore, we decided to run a Western blot against the his-tag attached to our insulin protein to see if our promoter worked and to get clarity about the results of our GFP experiment. The results of our Western blot, however, did not confirm that our sensor and actuator device worked to produce insulin. The Western blot showed no difference between our experimental sample of cells treated with 2% glucose sample and a negative control that had 0% glucose and 2% raffinose to keep the carbon source constant (Figure 3). We chose a 0% glucose treatment as the negative control because the cells would not be induced, and again we chose 2% glucose as our positive control because of the previous iGEM team’s report.4 However, the 2% glucose lane showed no sign of protein, and we concluded that the Western blot gives no evidence that the device works. The Western blot also does not invalidate the results of the GFP test, giving us no reason to believe that our T2A peptide was mis-sequenced.

see figure 3.

Western blot image for insulin protein in induced and non-induced yeast cells. A 10 kDa prestained protein ladder was run in the first lane, cell lysates from induced yeast was run in the second lane, and cell lysates from non-induced yeast cells was run in the third lane. Induced yeast were cultured overnight in 2% glucose + YPD and non-induced cells were cultured overnight in 2% raffinose + YPD.

V. Discussion

Errors and Future Experimental Revisions

Our results are inconclusive about the nature of our promoter. We have reason to believe that our promoter does not work as intended, as seen by the two measurements that did not show differences in insulin production or GFP expression at higher levels of glucose (see figures 2 and 3). However, both of our tests lacked a true positive control, which does not yet allow us to fully rule out that we made errors in our experimental procedure. For our GFP expression test, we should have used a CMV inducible promoter linked to GFP expression that would have constitutively expressed GFP. With such a positive control,we would have known our plate reader assay was working. In our Western blot, an ideal positive control would have been to include a single chain insulin protein attached to a His-tag to confirm that our Western Blot was working correctly to detect our desired protein.18 Although it was unlikely that the transfer failed because we did visualize a ladder on our final image, we could have used a concentration of antibody that was too low for detection.18